Parenteral pimaricin as treatment of systemic infections

ABSTRACT

An antifungal composition suitable for parenteral administration to a mammal includes an amount of pimaricin or an antifungal derivative thereof that is effective to inhibit the growth of a fungal infection in a mammal; a pharmaceutically acceptable dipolar aprotic solvent; and a pharmaceutically acceptable aqueous secondary solvent. The composition can be used in methods of preventing or treating a systemic fungal infection in a mammal. The composition can be prepared by dissolving pimaricin or an antifungal derivative thereof in the pharmaceutically acceptable dipolar aprotic solvent; adding to the solution a pharmaceutically acceptable aqueous secondary solvent; and in a preferred method, by subsequently lypohilizing the composition, whereby a dry, shelf-stable composition is produced. This dry composition can be reconstituted into an aqueous solution suitable for parenteral administration.

BACKGROUND OF THE INVENTION

The present invention relates to a drug formulation that is useful forthe treatment and suppression of systemic infections, for example thosecaused by Aspergillus and Fusarium species.

Disseminated fungal infections constitute one of the most difficultchallenges for clinicians caring for patients with hematological cancer(1). While the incidence of hematogenous candidiasis has beensignificantly reduced with the introduction of fluconazole prophylaxis,the opportunistic molds have became the leading cause of infectiousmortality in this patient population (2). Aspergillosis clearly remainsthe most common mold infection in patients with hematological cancer.However, new opportunistic pathogens have now emerged as a cause oflife-threatening infection worldwide. The most frequently reported ofthese pathogens is Fusarium (3-7). Infection with Fusarium is associatedwith a very high mortality and is typically refractory to amphotericinB. Since infection with this organism may mimic aspergillosis, patientsare usually treated with Amphotericin B (AMB), an agent with pooractivity against Fusariosis. In addition, the airways are the mostcommon primary site of inoculation and infection and are almost alwaysinvolved in disseminated disease (3-7). Hence, any drug with goodactivity against Fusariosis (particularly if it is also active againstAspergillosis) that could be given parenterally and also throughaerosolization or nebulization will significantly improve ourtherapeutic armamentarium.

In addition to being ineffective against Fusariosis, Amphotericin B, thefirst-line treatment for documented or suspected systemic moldinfections carries with it common (>75% of treated subjects),substantial and frequently dose-limiting nephrotoxicity, requiring attimes hemodialysis. The acute infusion-related adverse events (severeshaking chills, fever, nausea, vomiting, headache) are quite troublesometo patients. Other serious side effects, such as cardiac arrhythmias,bone marrow suppression, neuropathies, and convulsions are alsoencountered with the use of AMB, although less frequently (8). Theintroduction of liposomally encapsulated AMB was anticipated to improvethe control of systemic fungal infections (9,10). Its administrationchanged the drug's biodistribution, allowing significantly higher dosesto be delivered with (hopefully) better anti-fungal effects, withoutencountering serious nephrotoxicity (11-13). In spite of an increasedrenal tolerance to liposomal AMB compared with the parent drug, this newformulation has several limitations, including its high cost (presentlyaround $800 per day) which has limited its use, its toxicity profilewhich is identical to that of Amphotericin B (except for the kidneytoxicity) and the fact that there is no evidence that this new drugformulation has actually improved the ultimate control rate of seriousmycotic/mold infections. Liposomal AMB has recently received federalapproval for routine clinical use in the U.S.

The only important clinically available alternative to AMB for thetreatment of systemic mold infections is itraconazole (Sporinox™) (13,14, 15). This agent is presently available exclusively as an oralpreparation that is only erratically absorbed from the intestinal tract,yielding variable plasma concentrations with highly unpredictableanti-fungal activity (13) and has little or no activity againstFusarium. This bioavailability problem is particularly difficult tomanage in bone marrow transplant (BMT) patients who are at highest riskfor invasive mold infections. Such patients typically have severemucositis that interferes with their ability to swallow the itraconazolecapsule and also impairs the already erratic intestinal absorption ofthe drug. In addition, these patients commonly receive antacids or H2blockers, both agents known to interfere with the absorption ofitraconazole.

Based on the above considerations, the development of an effectiveantimycotic agent with low normal organ toxicity, high bioavailability,predictable pharmacokinetics after parenteral administration, andactivity against both Fusarium and Aspergillus appears highly desirable.Pimaricin, or natamycin (FIG. 1) would fulfill the criterion of being aneffective anti-fungal agent, exerting significant activity againstmolds, particularly Fusarium and Aspergillus. It was first isolated in1955 from a strain of Streptomyces (15). Pimaricin exhibited a widerange of in vitro activity against fungi, yeast, and trichomonads (15,16, 17). The drug was found to have little or no toxicity after oraladministration, being virtually non-absorbable from the gastrointestinaltract (16, 17). However, the lack of solubility of pimaricin in varioussolvents, both aqueous and organic, compatible with human administrationhas severely restricted its use in clinical medicine. Pimaricin'smedical utilization is currently confined to the topical treatment ofcorneal fungal infections (18) and the prevention of such infections incontact-lens users. In contrast, pimaricin's prominent chemicalstability paired with its apparent lack of intestinal absorption andsystemic toxicity formed the basis for its FDA-approved use in the foodindustry, where it is used to prevent the proliferation of(aflatoxin-producing) molds (19).

A parenterally acceptable, nontoxic formulation of pimaricin would bepotentially beneficial not only for cancer patients, but also for othergroups of immunocompromised patients, e.g. those suffering from HIV andthose having recently undergone open heart surgery, all of which arecommonly targets for opportunistic infections.

Past attempts to solubilize pimaricin in vehicles that are safe forintravascular administration in humans have all failed, despite the hardwork by Stuyk and others (15, 16, 17). Thus, Korteweg and coworkersattempted to solubilize the drug by mixing it with a complexpolysaccharide (16). Although the water-solubility of this formulationincreased dramatically, its antifungal in vitro activity decreased toabout 1/3 of that of native natamycin. Further, this preparation iscomparatively toxic in experimental animals, and it was therefore deemedunsuitable for systemic parenteral administration in humans (15).

SUMMARY OF THE INVENTION

One aspect of the present invention is an antifungal composition that issuitable for parenteral administration to a mammal. The compositionincludes an amount of pimaricin or an antifungal derivative thereof thatis effective to inhibit the growth of a systemic infection in a mammal;a pharmaceutically acceptable dipolar aprotic solvent; and apharmaceutically acceptable aqueous secondary solvent. Suitable dipolaraprotic solvents include N,N-dimethylacetamide (DMA) and dimethylsulfoxide (DMSO). The aqueous secondary solvent can be, for example,water, saline solution, or dextrose solution. It can also be an aqueouslipid emulsion. Suitable aqueous lipid emulsions include those thatcomprise a lipid component that includes at least one vegetable oil andat least one fatty acid. In one particular embodiment of the invention,the lipid component comprises at least about 5% by weight soybean oiland at least about 50% by weight fatty acids. The lipids in thecomposition are preferably present in a form other than liposomes (e.g.,at least about 50% by weight of the lipid is not in the form ofliposomes, more preferably at least about 75%, and most preferably atleast about 95%).

Another aspect of the present invention concerns a method of preventingor treating a systemic infection in a mammal. The method comprisesadministering parenterally to a mammal a composition as described above,in an amount that is effective to inhibit the growth of a systemicinfection in the mammal. Although the present invention is especiallyuseful for preventing or treating systemic fungal infections, it canalso be used for prevention and treatment of systemic infections causedby other infectious agents that are sensitive to pimaricin in vivo, suchas viruses.

Another aspect of the present invention concerns a method of preparingan antifungal composition for internal use in a mammal, especially ahuman. This method includes the steps of dissolving pimaricin or anantifungal derivative thereof in a pharmaceutically acceptable dipolaraprotic solvent; and adding to the solution a pharmaceuticallyacceptable aqueous secondary solvent. In one preferred embodiment, themethod further includes the step of lyophilizing the composition,whereby the majority of the water and the aprotic solvent (e.g., morethan 50%, preferably more than 95%, and most preferably more than 99% byweight) are removed from the composition and a dry, shelf-stablecomposition is produced. This dry composition can be reconstituted intoan aqueous solution suitable for parenteral administration to a mammal,by adding to the dry composition a pharmaceutically acceptable aqueoussolvent. Suitable pharmaceutically acceptable aqueous solvents forreconstituting the composition include the known parenteral infusionfluids, such as saline solution and dextrose solution in addition todistilled water.

We have examined the available methods for solubilization and devisednontrivial procedures for solubilizing this agent for parenteral use: wehave dissolved it using an organic solvent as the primary vehicle, e.g.dimethylacetamide, and then followed with secondary cosolvents toincrease the drug's stable aqueous solubility, or alternatively, we havefollowed the primary solubilization step with a second aqueous solventfollowed by lyophilization to create a pimaricin solvate with minimalorganic solvent content, yet one that could be easily reconstitutedusing distilled water only. Employing a variety of chemical andbiological assays we showed that the resulting final pimaricinformulations are stable for several hours at room temperature, and thatthey retain full antifungal activity. We ultimately used one of theformulations in a canine model to demonstrate that the reformulatedpimaricin permits what has heretofore been impossible, namely safeparenteral (e.g., intravascular) administration with negligibletoxicity, yielding clearly fungicidal plasma concentrations for morethan six hours following the administration.

The present invention provides vehicles for the formulation of pimaricinthat are physiologically compatible with parenteral administration inman and domestic animals. The pimaricin formulations of the presentinvention are non-toxic and can be used for the parenteral treatment ofsystemic infections sensitive in vitro to this compound, such asinfections of Candida, Aspergillus, and Fusarium, to circumvent thevirtually nonexistent intestinal absorption of the drug. The inventionwill allow the introduction of pimaricin in clinical practice for thetherapy of systemic infections, such that the therapeutic outcome forpatients with systemic infections sensitive to the drug can be improved.

A high-pressure chromatography technique that allows the accuratedetermination of low concentrations of pimaricin in various solventsystems and in biological fluids. This patent also describes our in vivocanine model for studying the pharmacokinetics of pimaricin afterparenteral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Chemical structure of pimaricin as free drug.

FIG. 2: Stability of pimaricin in DMA alone at 4° C. (♦), and at RT (22°C.) (□), at a concentration of 100 mg/ml. The y-axis shows the fractionof drug remaining as percentage of control (i.e, startingconcentration).

FIGS. 3A and 3B: HPLC chromatogram of pimaricin in the HPLC assay. FIG.3A: Pimaricin extracted from an aqueous solution of 5 μg/ml. FIG. 3B:Pimaricin extracted from a plasma sample spiked to a concentration of 5μg/ml.

FIG. 4: Stability of pimaricin at 4° C., 22° C., 40° C., and 60° C. Thepimaricin formulation was in DMA-aqueous lipid emulsion prepared"fresh." "AUC" is the area under the curve of the pimaricin peak in thechromatogram. This represents drug concentration, but in this experimentit was not translated into a numerical drug concentration using astandard curve plotting AUC vs. drug concentration.

FIG. 5: Stability over 48 hours of the final solution for clinical use,maintained at RT after dilution to 1 mg/ml. The symbols refer to thefollowing solutions: Pimaricin/L/NACL: the lyophilized and reconstitutedsolution was diluted from 10 mg/ml to 1 mg/ml with NS. Pimaricin/L/D5:as above, but the secondary solvent was 5% dextrose instead of NS.Pimaricin/NACL: the DMA/Intralipid™ formulation was prepared fresh to aconcentration of 10 mg/ml as described, and the secondary solvent usedwas NS. Pimaricin/D5: The same DMA/Intralipid™ formulation as above,prepared fresh, but the secondary solvent was 5% dextrose instead of NS.

FIG. 6: Hemolytic effects of the DMA/DMSO/PEG/PG formulation without (◯)and with pimaricin ().

FIG. 7: Hemolytic effect of the freshly prepared DMA/aqueous lipidformulation without (□) and with pimaricin (Δ). Negative control was 10%aqueous lipid (Intralipid™) alone (◯), at a concentration comparable tothat when pimaricin was added to the vehicle at the concentrationindicated on the abscissa.

FIG. 8: Hemolytic effect of the DMA/aqueous lipid solution lyophilizedand reconstituted in double-distilled water without (▪) and withpimaricin (▴). Negative control was the 10% aqueous lipid (Intralipid™)alone (◯), at a concentration comparable to that when pimaricin wasadded to the vehicle at the concentration indicated on the abscissa.

FIGS. 9A and 9B: Pimaricin formulated fresh in DMA/aqueous lipid wasassessed for toxicity against the KBM-7/B5 cells (▴), and against HL-60cells (▪), using the MTT assay for 48 hours (FIG. 9A), and for 72 hours(FIG. 9B) as described in materials and methods.

FIGS. 10A and 10B: HPLC chromatograms of a plasma sample analyzed withthe HPLC assay. FIG. 10A: Plasma blank samples before the start ofinfusion. FIG. 10B: Sample from a dog injected with 5 mg/kg body weightof pimaricin. The drug was given over 1 hour iv and this blood samplewas obtained 5 hours after drug infusion was completed. The sample wasextracted and analyzed as described in the text.

FIG. 11: Dose linearity of pimaricin utilizing the established HPLCassay in the concentration range 100 ng/ml to 25 μg/ml.

FIG. 12: Comparative plasma concentrations during and after infusion ofpimaricin at 1 mg/kg, and 5 mg/kg in four beagle dogs. The samples weredrawn just before the end of the 60 min infusion and 5 hours after theend of infusion. The different numbers and symbols, respectively, referto the individual animals, and the 1 and 5 respectively refer to thedose of pimaricin administered per kg body weight.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The following abbreviations are used in this patent:

AMB; Amphotericin B.

ATCC; American Tissue Culture Collection, Rockville, Md.

BMT; bone marrow transplant.

DMA; anhydrous N,N,-dimethylacetamide.

DMF; Dimethylformamide.

DMSO; Dimethylsulfoxide.

FDA; U.S. Food and Drug Administration.

HAc; Glacial acetic acid.

HCl; Hydrochloric acid.

HPLC; High pressure liquid chromatography.

HL-60; Human myeloid leukemia cell line.

IMDM; Iscove's modified Dulbecco Medium (GIBCO, Grand Island, New York,N.Y.).

Intralipid™; Brand name of an aqueous lipid emulsion, made from soy beanoil, and marketed for parenteral nutrition by Clintec.

KBM-7/B5; Human myeloid leukemia cell line.

MeOH; Methanol.

MIC; minimum inhibitory concentration.

MTT; 3,[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium-bromide.

NCI; National Cancer Institute.

NS; Normal saline (150 mM NaCl).

PEG; Polyethylene glycol-400.

PG; Polypropylene glycol/1,2-propylene diol.

RT; Room temperature (22° C.)

SDS; Sodium dodecyl sulphate.

The present invention involves solubilization of pimaricin inpharmaceutically acceptable liquid vehicles, such that the drug remainschemically stable and can be administered intravascularly without unduetoxicity from undissolved drug and/or from the solvents at drug dosesnecessary to obtain clinically significant antibiotic effects.

Pimaricin is available from Gist-Brocades N.V. (Netherlands) and SigmaChemical Co. (Saint Louis, Mo.). Pimaricin optionally can be used incompositions of the present invention in the form of one of itsantifuingal derivatives, such as a salt of pimaricin (e.g., an alkalisalt or an alkaline earth salt).

We have investigated N,N-dimethylacetamide (DMA), DMSO, glycerol,1,2,-propylene-diol (PG), and polyethylene glycol-400 (PEG) as primarysolvents that would be miscible in secondary solvents, examples of whichare normal saline, dextrose in water (5% or 10%), and an aqueous soybean lipid emulsion (Intralipid™). These solvents are examples ofvehicles in which pimaricin could be suitably solubilized, yet be safefor human administration, alone or in combinations with other drugs. Thesolubility of pimaricin in individual solvent vehicles is shown in Table1 below.

The described vehicles can be utilized to dissolve pimaricin inconcentrations ranging from 1 to more than 100 mg/ml. This range shouldcover the administration of doses necessary to yield active antibioticconcentrations in vivo that are effective to eradicate systemicinfections sensitive to this drug.

The objective of this invention includes the parenteral (e.g.,intravascular) administration of pimaricin to improve the control ofsystemic infections that are sensitive to this agent. As a paradigm forsuch infections, we will use various molds and other fungal organisms.This use of pimaricin as a parenteral agent has not been previouslyinvestigated in the practice of medicine, although the drug has welldocumented anti-fungal properties in vitro (15-17).

Virtually no pimaricin is absorbed through the intestinal tract afteroral administration, making it impossible to even investigate its use asan oral antibiotic against systemic infections. Parenteraladministration would therefore be the logical approach to evaluatepimaricin as therapy for deep-seated, systemic fungal infections.Unfortunately, the drug has an exceedingly low solubility in mostphysiologically acceptable solvents that would be compatible withintravascular administration in man (17).

Our present studies, which are based on the principle of cosolvency (20,21), show that the composite diluent vehicles we propose for use willsolubilize pimaricin without destroying its antifungal properties.Further, the preferred vehicles are nontoxic and safe for administrationin large animals (beagles) and should be acceptable for humanadministration in the proposed concentrations and total doses to beutilized; indeed, DMA, DMSO, and PG have been used for solubilization ofvarious pharmacologically active agents used in man (22-24). Theparenteral administration of PEG has been studied in detail in a simianmodel (25), and PEG has subsequently been used clinically as a(covalently bound) carrier of L-Asparaginase in the treatment oflymphocytic leukemia and lymphoma (26). DMSO is also extensively used asa cryoprotective agent for low-temperature storage of human bone marrowand peripheral blood derived hematopoietic stem cell preparations to beused for transplantation after high-dose chemotherapy (27-30). Noserious adverse effects have been experienced from the use of thesevehicles. The clinical use of normal saline, dextrose in water (5-70%),and aqueous lipid emulsion are well established means to alter the fluidand electrolyte balance and to supply parenteral nutrition. Normalsaline and dextrose in water are extensively used to dilute variousmedications for parenteral use. However, the aqueous lipid emulsion hasnot yet found wide-spread use as a pharmaceutical diluent, although thisuse has been mentioned (31).

The data obtained in our canine model demonstrate that the proposedpimaricin formulations, that is, those that allow parenteral treatmentof systemic infections, will provide superior bioavailability. After aone-hour i.v. infusion the plasma concentrations clearly reach, and foran extended time remain in, the fungicidal range as established by ourin vitro studies of antifungal activity against Candida spp.,Aspergillus spp., and Fusarium spp. Specifically, our novelpimaricin/DMA/lipid solution is chemically stable and simple to handleat RT. It provides reliable and easily controlled dosing with 100%bioavailability. The addition of a lyophilization step virtuallyeliminates the organic solvent, DMA, from the final clinical "workingsolution", and it should abolish the potential for adverse reactionsrelated to the DMA, and minimize the possibility for a potentiation of(hepatic) side effects from the combination of DMA and pimaricin. Thisadded step should therefore assist in maximizing patient safety afterdrug administration.

In cancer patients, the access to parenteral pimaricin will beparticularly important, since their intestinal absorption is oftenperturbed after chemotherapy, aggravating the already erratic intestinalabsorption of various medications. The parenteral route will also makeit possible to circumvent unpredictable first-pass metabolic effects inthe liver, well known to alter the bioavailability of numerouspharmacologically active agents after oral dosing (32). Further, theavailability of pimaricin for effective and reliable systemicadministration will for the first time make it possible to clinicallycompare the activity of pimaricin against that of "the gold standard",AMB, for the treatment of systemic mycoses.

In summary, certain infections in immunocompromised patients, e.g. thosecaused by various molds, particularly Fusarium, may be eradicated bypimaricin. In fact, pimaricin may be the only effective drug for thetreatment of Fusariosis, since this infection typically is resistant toAMB. The design of a nontoxic, pharmaceutically acceptable, watermiscible, parenteral formulation of pimaricin eliminates the risk oftreatment failure from the suboptimal bioavailability of oral pimaricin.The addition of a lyophilization step in the preparative procedure willcreate a pimaricin solvate with minimal DMA content. This will reducethe risk of adverse effects related to the vehicle's organic component.

The following examples are presented to describe the preferredembodiments and utilities of the present invention, but they are notintended to limit the invention to these aspects, unless otherwisestated in the claims appended hereto.

EXAMPLE 1 Pimaricin Formulations Acceptable for ParenteralAdministration

The objectives of this experiment were to design formulations ofpimaricin that are acceptable for parenteral administration, tocalculate the necessary solubility/stability needed to accomplish thisgoal, and to evaluate our ability to make such preparations with a highpressure liquid chromatographic (HPLC) technique.

METHODOLOGY Calculation of the Desired Solubility

We have calculated a relevant solubility range for pimaricin byextrapolation from known values for AMB. AMB is presently the onlypolyene antibiotic that is FDA-approved for parenteral use. Thecurrently utilized AMB regimens typically prescribe a daily dose of0.6-1.0 mg/kg body weight as free AMB or 5-6 mg/kg body weight forliposomally-complexed drug (11). We have assumed that a clinically safemaximum infusion rate for pimaricin is 2-3 ml/min over 60-120 minutes,thus arriving at peak plasma concentrations in the range of 3-15 μg/ml(4.5-20 μM). Such concentrations may be necessary if pimaricin treatmentis to be successful, since AMB and pimaricin on a molar basis have asimilar concentration vs. activity range in vitro (AMB about 0.3-10 μM,and Pimaricin about 3-20 μM). Therefore, the anticipated daily pimaricindose would be around 1.0-5.0 mg/kg body weight. If this dose weredissolved at a concentration of 1-5 mg/ml, a 50-100-fold increase overthe established aqueous solubility of 25-50 μg/ml at RT would berequired (17).

Enhanced Solubility in Physiologically Acceptable Solvents

Pimaricin solubility was determined in several individual vehicles.Briefly, a known amount of the drug, as a powder (different lots ofpurified drug were obtained from Gist-Brocades N.V., Netherlands, andfrom Sigma Chemical Company, St. Louis, Mo.), was equilibrated in therespective solvent at RT (22° C.) over 1-4 hours. An aliquot was thenremoved and diluted in MeOH prior to HPLC at predetermined times. Basedon the pimaricin solubility in these particular vehicles, we thenattempted to enhance the (stable) solubility by mixing differentsolvents according to the principle of cosolvency (20, 21). Severaldifferent solvent systems were evaluated relative to the above estimatesof necessary solubility to arrive at a clinically relevant optimal stockformulation. This stock formula would then be diluted with a "finalsolvent" to yield the complete working formulation with a pimaricinconcentration that could be infuised parenterally without problem. Forthe final solvent we used the commonly utilized parenteral infusionfluids, such as normal saline, dextrose in water (5% or 10%), or aparenterally acceptable aqueous lipid emulsion (e.g. Intralipid™ orLiposyn II™ (Abbott)), all of which are readily available and approvedfor parenteral administration.

HPLC Assay

A most accurate and sensitive detection system for low concentrations ofpimaricin in solution, both protein-containing and protein-freemixtures, is an HPLC assay utilizing absorbance detection with avariable wave length detector operating in the u.v. spectrum at 293 nm,a value chosen on the basis of the inherent absorption maxima of thepimaricin molecule (17).

We tested this hypothesis using a liquid chromatographic system equippedwith an LDC 4000™ multi-solvent delivery system and a Waters™ system717plus Autoinjector™. The absorbance detector was a LDC 3100 variablewave length detector in sequence with an LDC model CI 4100 fullycomputerized integrator. The column used was a Whatman EQC™ 10 μI 125AC18 column (4.6 mm i.d.×21.6 cm) (Whatman Inc. Clifton, N.J.). Themobile phase system was an isocratic mixture of MeOH (47% v/v),tetrahydrofuran (2% v/v), and NH₄ -acetate (0.1% w/v) made up to 100%with double-distilled water. All chemicals were HPLC grade unlessotherwise indicated. The flow rate was 1.5 ml/min and the recorder'schart speed was 5 mm/min, modified from (33).

RESULTS AND DISCUSSION Pimaricin Solubility

Several strategies were evaluated to solubilize pimaricin inwater-miscible physiologically acceptable vehicles that would becompatible with human administration. The examined candidate solventsincluded castor oil, DMA, DMSO, PEG, and PG, in addition to the aqueoussolvents HAc, NS, 5% dextrose in water and an aqueous soy bean emulsion(Intralipid™). HAc and DMA were the best primary solvents, followed byDMSO, whereas pimaricin as expected was insoluble in most of the aqueoussolvents. Only with HAc and DMA did we reach a solubility in excess of10 mg/ml. Further, although pimaricin could be dissolved in HAc and DMAto at least 100 mg/ml, it started degrading already within a few hoursin solution (FIG. 2). Stabilizing the pimaricin once dissolved in DMAwas then addressed with a cosolvency approach (20, 21). Numerouscosolvent combinations were investigated; the composite organic systemof DMA/DMSO/PEG/PG appeared to work well, but it did still only allowpimaricin to be dissolved at a final concentration of about 10 mg/ml.This composite vehicle did not allow stable solubilization of pimaricinfor more than a few hours. When NS or 5% dextrose in water was added,significant degradation rapidly took place. In contrast, a differentpattern was recorded when a lipid-containing cosolvent was utilized.When HAc was used as the primary solvent, the best secondary solventsappeared to be DMA, DMSO or Intralipid™.

HPLC Assay

Two examples of pimaricin chromatograms from the HPLC assay are shown inFIGS. 3A and 3B. In FIG. 3A the drug was analyzed in the aqueousDMA-Intralipid solvent, and in FIG. 3B it was extracted from humanplasma that had been spiked with 5 μg/ml prior to extraction asdescribed above. The retention time under the above conditions was9.8-10.8 min, and the assay was linear from 100 ng/ml to 25 μg/ml inprotein-free solutions, i.e. the various solvent systems utilized in theformulation-feasibility and -stability studies, and from about 50 ng/mlto 1 mg/ml for protein-containing solutions (plasma samples). This assayconsistently yielded high recovery, accuracy and a lower sensitivitylimit of about 10 ng/ml. The technique was standardized and used withoutmodifications for the studies of both stability and pharmacokinetics.

EXAMPLE 2 Solubility and Stability Studies of Various Formulations

The objectives of this experiment were to: (1) design stable pimaricinformulations that are suitable for parenteral administration; (2)establish the chemical and physical stability of pimaricin in the novelvehicles; (3) establish the solubility of pimaricin in these vehicleswhen mixed with NS, dextrose in water, and Intralipid™; and (4)investigate the in vitro properties of these formulations; i.e. theirosmolarity, hemolytic potential, and cytotoxicity, to show that they areappropriate for the intended purpose.

METHODOLOGY Solubility Studies

An excess amount of pimaricin as a solid powder was added to castor oil,DMA, DMSO, PEG, and PG at RT. Each mixture was placed in a darkenvironment and checked visually for up to 4 hours for evidence ofsolubilization. Samples of 1 ml were taken at various time intervals,and filtered through a 0.45 μm PTFE membrane filter fitted to a syringeassembly (Whatman Inc.), and after appropriate dilution, the pimaricinconcentration was determined by HPLC.

Stability of the Various Pimaricin Formulations

To study the physical and chemical stability of the various parenteralformulations, three sets of experiments were performed:

(a) Pimaricin was dissolved at a concentration of 100 mg/ml in DMA("stock solution") and incubated at 4° C., at 22° C. and at 40° C. Weanalyzed the drug concentration by HPLC in samples taken immediatelyafter solubilization and after gradually increasing time intervals of upto 48 hours.

(b) The pimaricin-DMA stock solution was diluted with PEG/water (1:1:1,v:v:v, DMA:PEG:water), or PG/DMSO (1:1:1, v:v:v), or PG/DMSO/PEG(1:1:1:1, v:v:v:v), or aqueous lipid emulsion (1:10 and 1:100, v:v,DMA:Intralipid™), to yield pimaricin concentrations ranging from 1-10mg/ml.

(c) The DMA-pimaricin mixture was diluted in NS or 5% dextrose to a drugconcentration of 1 mg/ml.

(d) The pimaricin-HAc mixture was blended with DMSO and Intralipid™, ordirectly in Intralipid™.

The various formulations were analyzed by HPLC immediately after mixing,then hourly for 8 hours, and then at gradually increasing time intervalsup to several weeks, depending on the rate of degradation in therespective solvent system.

The solubility of the drug differed markedly between different solvents(Table 1). Only DMA and HAc, which provided the highest solubility wereconsidered for extended studies as primary solvents.

                  TABLE 1                                                         ______________________________________                                        Solvents Tested for Solubilization of Pimaricin                                        Time Allowed to                                                                           Maximum Solubility                                       Formulation                                                                            Solubilize (hr)                                                                           (mg/ml)       Vehicle                                    ______________________________________                                        1        4            2            DMSO                                       2        4            10           DMA                                        3        6           100           DMA                                        4        4           0.078         PG                                         5        <0.2        >300          HAc                                        6        4           N/S           Castor oil                                 7        4           N/S           PEG 400                                    8        4           N/S           Intralipid                                 ______________________________________                                         (N/S indicates that pimaricin was not soluble in that solvent.)          

To lower the DMA concentration in the final stock- and use-formulationswithout adversely affecting the drug's shelf life, we investigatedlyophilization as part of the preparation of a completepimaricin/DMA/aqueous lipid-solvate vehicle.

Osmotic Pressure Measurement

Osmotic pressures were measured with a micro-osmometer model 3MOplusosmometer (Advanced Instruments Inc., Needham Heights, Mass.). Theinstrument was calibrated using Advans™ intrinsic calibration standards(Advanced Instruments Inc.) over a range of 500-2000 mOsm/kg. The testsolution was placed in a disposable cuvette from the test kit, and theosmotic pressure readings were recorded after equilibration in units ofmOsm/kg. Triplicate measurements were carried out for each vehicle(without pimaricin), and six measurements were done with pimaricinadded.

We used a two-tailed t-test to evaluate the differences in osmoticpressures of the various vehicle formulations with and without theaddition of pimaricin (34). The difference between the means of the twogroups was to be considered significantly different for P≦0.05.

Hemolysis Studies in vitro

We employed the procedure of Parthasarathy et al to examine thehemolytic potential of a few selected preparations (35), and the LD₅₀values of the various formulations were constructed as described.Briefly, heparinized blood was mixed with an equal volume of Alsever'ssolution. This mixture was washed twice in PBS, and a 10% (v/v)erythrocyte/PBS solution was then prepared and mixed with increasingamounts of the complete solvent system with or without the addition ofpimaricin. These mixtures were then incubated for 4 hours at 37° C. Atthe end of the incubation, the cells were pelleted at 10,000×g in anEppendorff™ centrifuge, and the release of hemoglobin in the supernatant(i.e. hemolysis) was spectrophotometrically determined at 550 nm.Maximum lysis was measured against a reference solution of erythrocytesthat had been completely lysed by hypotonic shock. The hemolyticpotential of three of the complete formulations was evaluated asdescribed (35), and the data were plotted as the fraction of healthycells versus ln (natural logarithm) (total volume percent). Total volumepercent was defined as the volume percent of the vehicle in the mixtureafter dilution with blood. This was done in an attempt to simulate thedilution of the respective drug formulation in the bloodstream afterparenteral administration. Healthy erythrocytes were defined as thosecapable of retaining their hemoglobin intracellularly after mixture withthe various pimaricin formulations (35).

In Vitro Cytotoxicity of Pimaricin

The cytotoxic potential of selected solvent systems with and withoutpimaricin was determined against the two human myeloid leukemia celllines HL-60 (36) and KBM-7/B5 (37, 38), using a modification of thepreviously published MTT assay (39, 40). Briefly, HL-60 or KBM-7/B5cells in Iscove's modified Dulbecco medium (IMDM) supplemented with 10%fetal bovine serum were incubated for 60 min at 37° C. with the completevehicles (a: DMA/PG/DMSO/PEG in ratios 1:1:1:1, v/v, and b:DMA/lntralipid™, 1:10, v/v, or c: HAc/DMSO/Intralipid™, 2:6:3, v/v) atincreasing concentrations of the vehicle (0.5%, 1.0%, 2.0%, 3.0%, and10%, v/v) with or without pimaricin. At the end of the 60 min incubationthe cells were washed in ice-cold PBS and resuspended in IMDM with 10%fetal bovine serum at 37° C. Twenty-four hours later 25 μl MTT solution(5 mg/ml) (Sigma Chemicals, St. Louis, Mo.) was added to each sample,and following an additional 2 hours of incubation at 37° C., 100 μlextraction buffer was added [extraction buffer: 20% (w/v) SDS dissolvedto saturation at 37° C. in a solution of DMF and deionized water (1:1);pH 4.7]. After incubation overnight at 37° C., the optical densitieswere measured at 570 nm using a Titer-Tech™ 96-well multi-scanner™,against extraction buffer as the calibrating blank. The cytotoxicity wasdetermined as the colorimetric difference between the samples exposed tosolvent ±pimaricin as above and the background reactivity of cells thathad been incubated in parallel in PBS alone. All determinations wereperformed in triplicate (39, 40).

RESULTS AND DISCUSSION Equilibrium Solubility Determinations andStability Studies in Various Solvent Vehicles

A maximum equilibrium solubility of pimaricin of >100 mg/ml was achievedin DMA after 4 hours at RT. The drug formulations in castor oil, DMSO,PEG-400 and PG achieved considerably lower equilibrium concentrations(Table 1). The latter solvents neither provided an acceptable solubilitynor chemical stability of the dissolved drug, and these vehicles weretherefore not considered for further studies. Once a pimaricinsolubility of 100 mg/ml was reached in anhydrous DMA and HAcrespectively, the drug started degrading with a loss of approximately5-10% over the subsequent 3-4 hours. The drug was more stable when PEGwas used as a secondary solvent, but again drug degradation began withinanother few hours at RT. At 4° C. the drug was more stable, butdegradation was still apparent within 8 to 12 hours.

The temperature-dependent stability of solubilized pimaricin in thedifferent solvent systems was studied as follows: The drug was dissolvedin DMA at 100 mg/ml, and different aliquots were stored at 4° C., at 22°C., and at 40° C. Immediately after solubilization and at variousintervals up to 48 hrs later, aliquots from the different samples wereanalyzed by HPLC. The drug samples stored at 4° C. and at 22° C.degraded slower than those stored at higher temperatures: at 40° C. thepimaricin started degrading within 1 hour after the start of incubation,and at RT there was a loss of 5-10% in the first four hours.

When the 20% aqueous lipid emulsion (Intralipid™) was used as asecondary solvent, a different stability pattern was recorded; when thepimaricin concentration was adjusted to 1-10 mg/ml by dilution with 20%Intralipid of the DMA-pimaricin and the HAc-pimaricin stock solutions,the drug was stable for more than 7 days (FIG. 4).

The major fraction of the organic solvent, DMA, was removed bylyophilization of the pimaricin/DMA/aqueous lipid complex to create asolvate that was stable yet easily reconstituted by adding onlydouble-distilled water under gentle agitation without any appreciableloss of anti-fungal efficacy. Indeed, within a few minutes afteraddition of distilled water to the solvate, the drug was reconstitutedat 1-10 mg/ml, with only trace amounts of the organic solvent remaining.This reconstituted pimaricin formulation retained an anti-fungalefficacy that was equivalent to that of the freshly prepared DMA/aqueouslipid formulation when assayed in vitro (see below under Example 3).This reconstituted formulation was also stable at 4° C. for more than 2weeks. The lyophilized pimaricin formulation remained stable (by HPLC)for more than four months at 4° C. This preparation could still bereadily reconstituted to 10 mg/ml within a few minutes with distilledwater, with retention of full anti-fungal activity in vitro (see Tables3 and 4 below).

We further simulated a final clinical use-formulation with a pimaricinsolution of 1 mg/ml by diluting the 10 mg/ml-formulations (preparedfresh with DMA/Intralipid or after lyophilization/reconstitutionrespectively) with 5% dextrose or NS. FIG. 5 shows the respectivestability at RT of these "use-formulations". Similarly, when HAc andDMSO were used as the primary solvent system prior to mixing withIntralipid and followed by lyophilization, the majority of the organicsolvent, here DMSO, was removed and the result was a stable lipid-basedsolvate, that could be easily reconstituted to 10 mg/ml under gentleagitation after the addition of distilled water. This reconstitutedformulation was also stable for more than 24 hours at RT assessed byHPLC.

Osmotic Pressure

It is desirable that a parenteral formulation of a pharmacologicallyactive agent be isosmotic to blood. A hypertonic delivery system can beutilized if the drug/solvent is infused through a (central) venouscatheter and gradually diluted in a large blood volume. The osmoticpressure of the various formulations is shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Osmotic Pressures of Various Vehicles with and without Pimaricin                                         Osmotic pressure                                   Solution             n     Osm/kg                                             ______________________________________                                        Water                3      3                                                 Normal saline        3     233                                                5% dextrose in water 3     286                                                Blood, human         6     280-295                                            DMA:PEG:PG           3     4492                                               Pimaricin in DMA:PEG:PG                                                                            3     4732                                               Intralipid           3     340                                                DMA:Intralipid (1:10, v/v)                                                                         3     2067                                               Pimaricin in DMA:Intralipid                                                                        3     1930                                               (1:10, V/V, fresh)                                                            DMA:Intralipid       3     157                                                (1:10, lyophil.-reconstit.)                                                   Pimaricin (1 mg/ml) in DMA:Intralipid                                                              3     208                                                (1:10, lyophil.-reconstit.)                                                   Pimaricin (25 mg/ml) in DMA:Intralipid                                                             3     243                                                (1:10, lyophil.-reconstit.)                                                   ______________________________________                                         ("n" represents the number of independent determinations.)               

The DMA-stock formulation with or without pimaricin was very hypertonic;its osmotic pressure was more than 1,900 mOsm/kg, as compared with280-295 mOsm/Kg for human blood. The DMA/PG/DMSO/PEG and DMA/PEGsolvents were almost as hypertonic. In contrast, the DMA/Intralipidpreparation was closer to isosmotic when reconstituted afterlyophilization. Similarly, the lyophilized/reconstitutedHAc/DMSO/Intralipid™ vehicle was also close to isosmotic. Addingpimaricin to the respective vehicles did not appreciably change theirosmolarity (P>0.05).

Hemolysis

As shown in FIGS. 6-8, the formulations studied showed similar trendsfor hemolysis with the addition of pimaricin. The pimaricin dependentlysis was notable at concentrations exceeding 40 μg/ml for the compositeorganic solvent and at ≧50 μg/ml for the freshly prepared DMA/Intralipidformulation and at ≧60 μg/ml for the lyophilized-reconstitutedDMA/aqueous lipid formulation. The drug-specific hemolysis was highlyreproducible between different experiments, as was the internal rankingbetween the various solvent systems between the different experiments.The detailed data for the different vehicles with and without pimaricinare summarized in FIGS. 6-8. LD₅₀ values can be deduced from thisinformation. The DMA/Intralipid™ "fresh" formulation had a significantlylower hemolytic potential than the DMA/PEG/PG/DMSO composite organicvehicle. Further, the hemolytic potential of the lyophilizedDMA/Intralipid formulation was significantly lower than that of thefreshly prepared DMA/aqueous lipid formulation for all pimaricinconcentrations from 1 μg/ml up to 100 μg/ml. Finally, pimaricin-inducedhemolysis in all of the tested vehicles was significantly lower(>10-fold ) than that observed for various AMB formulations (LD₅₀ valuesin the range of about 4-5 μg/ml) under similar experimental conditions(41).

In Vitro Cytotoxicity of Pimaricin

The HL-60 and KBM-7/B5 myeloid cells were exposed to the selectedvehicles at increasing volume ratios with or without the addition ofincreasing drug concentrations. The cytotoxicity of each formulation wasthen assayed in the MTT assay (39, 40). None of the examined solventsystems exerted any detectable toxicity against the cells in this assay(FIGS. 9A and 9B).

EXAMPLE 3 Antifungal Activity of Solubilized Pimaricin

The objective of this experiment was to critically evaluate the in vitroantifungal activity of pimaricin when solubilized in a few selectedvehicles using solution AMB as the reference solution.

METHODOLOGY

The antifungal activity of pimaricin was compared with that ofamphotericin B utilizing a previously described assay (42). Briefly,serial dilutions of pimaricin and AMB were mixed in RPMI growth mediumwith L-glutamine and MOPS-buffer, pH 7.0 (Sigma Chemical Co., St. Louis,Mo.). The different strains of Candida, Aspergillus and Fusarium spp.were then added to the dishes. After incubation at 35° C. for 48-72hours the plates were evaluated for fungal proliferation. The usedfungal strains were obtained from the ATCC or isolated from patients,primarily at the MD Anderson Cancer Center.

The pimaricin concentrations in the used solutions were assayed inparallel with HPLC to assure the highest possible reproducibility of thedrug concentrations.

RESULTS AND DISCUSSION

sensitivity data are displayed in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        Sensitivity of Fungal Organisms Against Various                               Pimaricin Formulations                                                                                             F/D                                                             L/D    Rm-temp                                                                              (nata + lipid)                           Organism    Code       μg/ml                                                                             μg/ml                                                                             μg/ml                                 ______________________________________                                        Aspergillus fumigatus                                                                     6-2535     2      2      2                                        Aspergillus fumigatus                                                                     6-7784     2      2      2                                        Aspergillus niger                                                                         6-2165     2      2      2                                        Aspergillus fumigatus                                                                     6-5337-1   2      2      2                                        Fusarium moniliformi                                                                      M6306      2      2      2                                        Aspergillus flavus                                                                        6-4594-2   >16    >16    >16                                      Fusarium solanii                                                                          s-1184     2      2      2                                        Candida albicans                                                                          ATCC 64545 2      2      2                                        ______________________________________                                    

The organisms of Table 3 were prepared as specified in the methodologyin Example 3. "L/D" refers to a formulation where pimaricin wasdissolved to 100 mg/ml in DMA, then diluted to 10 mg/ml with 20%Intralipid, lyophilized and then stored for >4 months at 4° C., followedby reconstitution in normal saline to 10 μg/ml as "use-solution"."Rm-temp" refers to a formulation where pimaricin was prepared fresh inDMA and Intralipid (10 mg/ml), kept for one week at RT, and then testedfor its antifungal properties. "F/D (Nata+lipid)" refers to aformulation where pimaricin was freshly dissolved at 100 mg/ml in DMAand then diluted with 20% Intralipid to 10 mg/ml as a fresh use-solutionthat was diluted to final concentrations of <2 to 16 μg/ml as describedherein.

Table 4 reports the results of another similar experiment.

                  TABLE 4                                                         ______________________________________                                        Sensitivity of Fungal Organisms Against Various                               Pimaricin Formulations                                                                             Lipid +                                                                        DMA    Nata- Nata- AMP +                                                     (1:10)  lipid 1                                                                             lipid 2                                                                             DMSO                                 Organism    Code     μg/ml                                                                              μg/ml                                                                            μg/ml                                                                            μg/ml                             ______________________________________                                        Aspergillus fumigatus                                                                     6-2535   >16     2     2     0.125                                Aspergillus fumigatus                                                                     6-7784   >16     2     2     0.25                                 Aspergillus niger                                                                         6-2165   >16     2     2     0.03                                 Aspergillus fumigatus                                                                     6-5337-1 >16     4     4     0.5                                  Aspergillus flavus                                                                        6-4594-2 >16     >16   >16   1                                    Aspergillus fumigatus                                                                     6-209    >16     2     2     0.25                                 Aspergillus fumigatus                                                                     6-0960   >16     2     2     0.25                                 Aspergillus fumigatus                                                                     6-1886   >16     4     4     0.25                                 Aspergillus fumigatus                                                                     6-1261   >16     4     4     0.25                                 Aspergillus flavus                                                                        4-9044   >16     >16   >16   1                                    Aspergillus flavus                                                                        6-5337-2 >16     >16   >16   1                                    ______________________________________                                    

"Lipid+DMA" refers to freshly mixed DMA and Intralipid (1:10, v/v),which exerts no antifungal activity by itself. For "Nata-lipid 1" and"Nata-lipid 2," pimaricin was dissolved in DMA to 100 mg/ml then dilutedwith 20% Intralipid to 10 mg/ml "use-formulation." "Nata-lipid 1" refersto a formulation where pimaricin was dissolved as above, and afterdilution to 10 mg/ml using Intralipid, it was lyophilized. Thelyophilized material was refrigerated for 4 months, then reconstitutedin normal saline to 10 mg/ml and tested for antifungal activity."Nata-lipid 2" refers to a formulation where thepimaricin/DMA/Intralipid formulation was prepared as for Nata-lipid 1and lyophilized immediately, and was reconstituted and tested forantifungal activity three days later. "AMP+DMSO" refers to a formulationof Amphotericin B dissolved immediately prior to use in DMSO, to serveas a positive control.

The activity of pimaricin was similar to that of AMB. Most of theAspergillus and Fusarium spp. were sensitive to pimaricin, independentof the solvent system. Importantly, the DMA/Intralipid™ formulation thatwas lyophilized and reconstituted with distilled water only, retainedfull and stable anti-fungal efficacy, when assayed both after 3 days andafter more than 4 months at 4° C. All the Aspergillus strains, exceptfor A. flavus, had pimaricin MIC values in the 2-4 μg/ml (2.1-4.2 μM)range. The tested A. flavus was also sensitive to the drug, but with aslightly higher MIC value of 16 μg/ml (17 μM). All the tested strains ofFusarium and Candida spp. were sensitive to pimaricin in the range of2-4 μg/ml (Tables 2 and 3).

EXAMPLE 4 Quantitative Pimaricin Analysis in Plasma and Pharmacokineticsof iv Pimaricin

The objective of this experiment were:

(1) To show that the drug can be administered intravenously andrecovered from the plasma from experimental animals using a quantitativeextraction technique and HPLC assay; and

(2) To show that the pimaricin plasma pharmacokinetics after ivadministration of the DMA/20% aqueous lipid formulation in beagle dogsare appropriate for treating systemic microbial diseases, in particularFusariosis.

METHODOLOGY Quantitative Extraction of Pimaricin in Plasma

Canine plasma (0.2 ml) and human plasma (0.5 ml) were mixed with variousamounts of pimaricin (in <3% of the final volume), to yield a drugconcentration of 0.05-3.0 μg/ml (from a pimaricin stock solution inDMA/20% Intralipid™ at a concentration of 10 mg/ml). The drug wasextracted from plasma samples using a slight modification of the methoddescribed by Napoli et al (43). Briefly, 0.2 ml plasma was mixed with0.2 N HCl in MeOH (1:1, v/v), and after thorough mixing by a vortexmachine, the sample was extracted with three volumes of hexane. Thehexane was separated from the pimaricin by evaporation and the drug wasreconstituted in 200 μl of MeOH prior to HPLC (43). Pimaricin wasspectrophotometrically detected in the HPLC analysis as described aboveon page 14. The pimaricin recovery from human plasma spiked to apimaricin concentration of 10 μg/ml was calculated to be 91±5%, and fromcanine plasma it was estimated to be in the order of 85±4%. The assaywas linear in the interval from 50 ng/ml to at least 1,000 μg/ml.

Parenteral Pimaricin in Beagles: Experimental Protocol

For the pharmacokinetics experiment we elected to use beagle dogs, sincethese animals are exceedingly sensitive to the toxic adverse effects ofpolyene antibiotics, and particularly to the nephrotoxic effects ofthese agents. The pimaricin was formulated in DMA/Intralipid™ to a stockdrug concentration of 10 mg/ml, and then diluted with Intralipid™, sothe doses (1.0 mg/kg/day in two dogs and 5.0 mg/kg/day in two otherdogs) could be administered IV in a volume of 10 ml over 1 hour by pumpthrough a cephalic vein catheter. To assure reproducibility of theexperimental conditions, the infusions were staggered; one dog at eachdose level was started on two consecutive days. The investigation wasperformed in male beagle dogs weighing 10-14 kg. The animals were notanesthetized but were restrained in a hanging sling during the druginfusion, which was performed at the same time daily for 14 consecutivedays. EKGs were recorded and blood samples were obtained fordetermination of pimaricin concentrations prior to the drug infusion andat various times during and following the infusion on the first day andon the last day of drug infusion. Blood for analysis of liver and kidneyfunction, as well as for differential and complete blood counts, andplatelet counts, was obtained in the morning before the first druginfusion, and also on days 8 and 15.

All animals were allowed free access to food and water, but with somerestriction to space and mobility, since we were concerned thatparenterally administered pimaricin could be cardiotoxic and cause fatalarrhythmias in a fashion similar to that of AMB, another polyeneantibiotic.

The drug was administered through the cephalic vein with good tolerance.The cannula and tubing were carefully flushed with heparinized salineafter each injection to prevent clot formation and to prevent drug fromadhering to the catheter wall and thus interfering with the bloodsampling for routine chemistries and for the pharmacokinetic analysis.

Blood samples of 3 ml were drawn in heparinized tubes before druginfusion, and at 10, 30, 55, 65, 70, 80, and 100 min, and at 2, 4, 6,12, 18, and 24 hours after the start of the infusion. The blood wascentrifuged at 1,000×g for 10 min, and the plasma was separated andstored at -80° C. until assayed by HPLC.

RESULTS AND DISCUSSION OF THE DATA Pimaricin in Plasma and iv DrugPharmacology

The drug extraction with hexane and MeOH from plasma was essential toavoid interference from endogenous plasma components and to recover themaximum amount of drug. Chromatograms from blank plasma,pimaricin-spiked plasma, and one example of that obtained afterextraction of a plasma sample from the current pharmacokinetic study areshown in FIGS. 10A and 10B. The pimaricin retention time in this systemwas 9.8-10.8 min. The recovery of pimaricin with the above describedtechnique was 91±5% when human plasma was spiked in vitro with 10 μg/mlof drug. The assay was linear after drug extraction from plasma samplesin the range from 50 ng/ml to 1.0 mg/ml. The drug recovery from canineplasma was 85±4%, with an accuracy of 98% and a limiting sensitivity ofabout 10 ng/ml. A standard curve was prepared in the concentration rangefrom 100 ng/ml to 25 μg/ml (FIG. 11), and a good correlation wasobtained between the plasma pimaricin concentration and peak AUC value("AUC" refers to the area under the curve measurement that one gets asthe exact reading from the fluorescence detector. I can be translated todrug concentration using a standard curve:

    AUC=1.2209e+4+3.2994e+5x, r.sup.2 =1.00.                   (Eq. 1)

where e is the exponential function, x is the drug concentration that issought, and r² is the correlation coefficient for the linear regressionanalysis for the ideal curve obtained from the actual data points in theobservation interval.

The in vivo peak plasma pimaricin concentrations after iv administrationof the above formulation was plotted for the two dose levels at the endof the 1 hour infusion and 5 hours later (FIG. 12); the measuredconcentrations are all within the in vitro range of sensitivity for themajority of the examined fungal isolates (see Tables 2 and 3).

Animal Experiment

There were no clinically discernible cardiac arrhythmias assessedthrough clinical monitoring and serial EKGs before, during, andfollowing the pimaricin infusions, and neither was there any detectedimpairment of hepatic or renal function over the 14-day experiment(Table 5). Group A consisted of two dogs (1 and 2) which were dosed at1.0 and 5.0 mg/kg/day, respectively. Group B consisted of two dogs (3and 4) which were also dosed at 1.0 and 5.0 mg/kg/day, respectively.Doses were administered to Group A on days 1-14 and to Group B on days2-15. Samples were taken from Group A on day 0 (the day before treatmentstarted), day 8 (after the first seven daily injections but before theeighth), and day 15 (the day after the final treatment). Samples weretaken from Group B on day 1 (the day before treatment started), day 9(after the first seven daily injections but before the eighth), and day16 (the day after the final treatment).

                                      TABLE 5                                     __________________________________________________________________________    Serum chemistry values in beagles after daily intravenous infusions of        Pimaricin over a 14-day period                                                Group                                                                             Dog                                                                              Dose                                                                             Na K  Cl BUN                                                                              Creat                                                                            P  TP                                                                              Albu                                                                             DB                                                                              LDH                                                                              AST                                                                              ALT                                                                              TB                                                                              AP  GGT                                                                              Mg                       __________________________________________________________________________    Day 0 or Day 1 (baseline)                                                     A   1  1.0                                                                              139                                                                              4.5                                                                              105                                                                              13 0.8                                                                              3.9                                                                              5.9                                                                             3.4                                                                              0.0                                                                             518                                                                              84 52 0.3                                                                             195 1  1.3                      A   2  5.0                                                                              142                                                                              4.6                                                                              107                                                                              14 1.4                                                                              3.0                                                                              6.0                                                                             3.5                                                                              0.0                                                                             247                                                                              36 31 0.2                                                                              84 2  1.2                      B   3  1.0                                                                              148                                                                              5.7                                                                              112                                                                              21 1.2                                                                              4.8                                                                              6.5                                                                             3.9                                                                              0.0                                                                             467                                                                              40 38 0.2                                                                              88 1  1.8                      B   4  5.0                                                                              146                                                                              4.7                                                                              109                                                                              15 1.1                                                                              3.6                                                                              6.6                                                                             3.7                                                                              0.0                                                                             196                                                                              33 68 0.3                                                                             261 2  1.7                      Day 8 or Day 9                                                                A   1  1.0                                                                              143                                                                              4.9                                                                              110                                                                              18 0.7                                                                              3.8                                                                              5.6                                                                             3.0                                                                              0.0                                                                             597                                                                              63 32 0.1                                                                             174 5  1.9                      A   2  5.0                                                                              122                                                                              5.9                                                                               75                                                                              90 1.8                                                                              6.4                                                                              8.2                                                                             3.9                                                                              0.2                                                                             624                                                                              122                                                                              98 0.5                                                                             1093                                                                              12 3.4                      B   3  1.0                                                                              135                                                                              4.9                                                                              103                                                                              20 0.9                                                                              3.7                                                                              5.6                                                                             3.3                                                                              0.0                                                                             675                                                                              54 37 0.2                                                                              80 3  1.9                      B   4  5.0                                                                              132                                                                              3.8                                                                               98                                                                              24 0.9                                                                              3.0                                                                              5.5                                                                             2.7                                                                              0.1                                                                             527                                                                              59 232                                                                              0.2                                                                             911 15 1.7                      Study termination (day 15 or day 16)                                          A   1  1.0                                                                              143                                                                              5.1                                                                              113                                                                              20 0.8                                                                              3.6                                                                              5.4                                                                             3.1                                                                              0.0                                                                             345                                                                              54 33 0.2                                                                             109 3  1.8                      A   2  5.0                                                                              111                                                                              10.3                                                                              71                                                                              247                                                                              3.5                                                                              17.5                                                                             8.6                                                                             4.2                                                                              --                                                                              751                                                                              545                                                                              205                                                                              1.0                                                                             625 -- --                       B   3  1.0                                                                              144                                                                              5.2                                                                              111                                                                              20 1.1                                                                              4.6                                                                              5.5                                                                             3.5                                                                              0.0                                                                             211                                                                              33 34 0.1                                                                              56 3  2.0                      B   4  5.0                                                                              144                                                                              4.0                                                                              108                                                                              21 0.9                                                                              3.2                                                                              5.3                                                                             2.9                                                                              0.0                                                                              63                                                                              26 64 0.2                                                                             424 8  1.6                      __________________________________________________________________________

Animal 2 died on day 12 of the study. Blood was obtained and analyzed,with the exception of levels listed as (--), immediately post-mortem.Abbreviations used in the table have the following meanings. Magnesiumlevel indicated for animal 4 on day 9 is the average of two readings.

    ______________________________________                                        Na            sodium                                                          K             potassium                                                       Cl            chloride                                                        BUN           blood urea nitrogen                                             Creat         creatinine                                                      P             phosphorus                                                      TP            total protein                                                   Albu          albumin                                                         DB            direct bilirubin                                                LDH           lactic dehydrogenase                                            AST           serum aspartate aminotransferase                                ALT           serum alanine aminotransferase                                  TB            total bilirubin                                                 AP            alkaline phosphatase                                            GGT           gamma glutamyl transpeptidase                                   Mg            Magnesium                                                       ______________________________________                                    

                                      TABLE 6                                     __________________________________________________________________________    Hematologic values in beagles after daily intravenous infusions of            Pimaricin over a 14-day period                                                Group                                                                             Dog                                                                              Dose                                                                             PT PTT                                                                              Fibr                                                                             FDP                                                                              RET                                                                              WBC                                                                              HGB                                                                              HCT                                                                              MCV                                                                              PLT                                                                              Neu                                                                              Lym                                                                              Mon                                                                              Eos                                                                              Baso                      __________________________________________________________________________    Day 0 or Day 1 (baseline)                                                     A   1  1.0                                                                              6.0                                                                              13.5                                                                             420                                                                              neg                                                                              0.4%                                                                             39.1                                                                             12.8                                                                             37.3                                                                             68.0                                                                             246                                                                              92.2                                                                              3.1                                                                             4.1                                                                              0.2                                                                              0.0                       A   2  5.0                                                                              5.5                                                                              13.3                                                                             190                                                                              neg                                                                              0.1%                                                                             12.1                                                                             14.4                                                                             41.5                                                                             67.3                                                                             388                                                                              65.4                                                                             26.4                                                                             3.8                                                                              3.6                                                                              0.3                       B   3  1.0                                                                              6.6                                                                              15.8                                                                             330                                                                              neg                                                                              0.7%                                                                             12.9                                                                             16.4                                                                             49.1                                                                             69.9                                                                             544                                                                              73.5                                                                             18.8                                                                             4.9                                                                              2.4                                                                              0.2                       B   4  5.0                                                                              5.5                                                                              15.3                                                                             420                                                                              neg                                                                              0.4%                                                                              5.8                                                                             17.2                                                                             50.6                                                                             70.3                                                                             208                                                                              52.7                                                                             28.8                                                                             14.1                                                                             4.1                                                                              0.1                       Day 8 or Day 9                                                                A   1  1.0                                                                              6.0                                                                              12.3                                                                             390                                                                              neg                                                                              4.6%                                                                             21.9                                                                             11.6                                                                             35.0                                                                             71.0                                                                             346                                                                              80.0                                                                             10.4                                                                             7.1                                                                              1.8                                                                              0.2                       A   2  5.0                                                                              7.0                                                                              15.3                                                                             555                                                                              neg                                                                              2.8%                                                                             48.8                                                                             18.6                                                                             53.0                                                                             64.1                                                                             112                                                                              89.0                                                                              5.4                                                                             4.2                                                                              0.7                                                                              0.1                       B   3  1.0                                                                              5.3                                                                              12.8                                                                             230                                                                              neg                                                                              6.9%                                                                             15.2                                                                             12.6                                                                             37.9                                                                             70.3                                                                             358                                                                              65.2                                                                             23.6                                                                             6.9                                                                              3.7                                                                              0.2                       B   4  5.0                                                                              5.8                                                                              13.5                                                                             430                                                                              neg                                                                              7.2%                                                                             16.2                                                                             13.6                                                                             40.5                                                                             71.6                                                                             138                                                                              75.4                                                                             11.9                                                                             10.3                                                                             1.8                                                                              0.1                       Study termination (Day 15 or 16)                                              A   1  1.0                                                                              7.8                                                                              12.0                                                                             220                                                                              neg                                                                              2.1%                                                                             14.2                                                                             11.0                                                                             33.4                                                                             72.4                                                                             255                                                                              80.0                                                                             14.0                                                                             3.9                                                                              1.8                                                                              0.1                       A   2  5.0                                                                              10.8                                                                             23.5                                                                             330                                                                              neg                                                                              0.4%                                                                             81.6                                                                             19.6                                                                             53.2                                                                             62.0                                                                             202                                                                              96.0                                                                              2.0                                                                             1.0                                                                              0  0                         B   3  1.0                                                                              8.3                                                                              13.3                                                                             280                                                                              neg                                                                              0.9%                                                                             14.5                                                                             12.3                                                                             37.0                                                                             71.3                                                                             421                                                                              65.4                                                                             25.2                                                                             4.4                                                                              4.6                                                                              0.1                       B   4  5.0                                                                              8.3                                                                              13.0                                                                             310                                                                              neg                                                                              1.1%                                                                             19.4                                                                             11.6                                                                             35.1                                                                             72.4                                                                             152                                                                              76.6                                                                             11.7                                                                             8.4                                                                              3.0                                                                              0.1                       __________________________________________________________________________

As mentioned above, animal 2 died on day 12 of the study. Blood wasobtained and analyzed, with the exception of MCV which was calculated,immediately post-mortem. Abbreviations used in Table 6 have thefollowing meanings.

    ______________________________________                                        PT              prothrombin time                                              PTT             partial thromboplastin time                                   Fibr            fibrinogen                                                    FDP             fibrin degradation products                                   RET             reticulocytes                                                 WBC             white blood cell count                                        HGB             hemoglobin                                                    HCT             hematocrit                                                    MCV             mean corpuscular volume                                       PLT             platelet count                                                Neu             neutrophils                                                   Lym             lymphocytes                                                   Mon             monocytes                                                     Eos             Eosinophils                                                   Baso            Basophils                                                     ______________________________________                                    

We found mild signs of hemolysis in the form of a gradual lowering ofhemoglobin and hematocrit levels and a slight increase in reticulocytecounts during the study (Table 6). There was, however, no sign of bonemarrow suppression/toxicity assessed by the white blood cell count,platelet count, or fibrinogen levels or any of the coagulationparameters (see Table 6). (Normal values for various hematological andserum chemistry parameters are provided in reference 44.)

Our data demonstrate the successful design of pharmaceuticallyacceptable formulations of pimaricin, ones that are physiologicallycompatible with parenteral administration, with good tolerance andnegligible toxicity, as demonstrated in the canine model. Theintravenous infusion of one of the preparations in beagles providedplasma concentrations that reached and over many hours maintainedfungicidal pimaricin concentrations without any discernible untowardeffects on the animals' clinical performance or as detected byassessment of their hepatic or renal function during the 2-weekexperiment. It should be noted, that for this experiment we selected the"fresh" DMA/aqueous lipid formulation that had the highest concentrationof an organic solvent, DMA, to allow for the least favorable scenariowhen considering the potential for adverse influence of the solventsystem on hepatic and renal function, as well as on the hematopoieticand cardiovascular systems.

Our data obtained with several diverse formulations demonstrateconclusively that it should be feasible to introduce parenteralpimaricin in clinical therapy of systemic fungal infections includingfusariosis, with the predictable attainment of antibiotic activity, andwith a reasonable expectation of low normal organ toxicity. Theinclusion of a lyophilization step in the formulation proceduresignificantly increased the stability/shelf-life of the finalformulations. This step virtually eliminates the final use-preparation'scontent of the organic solvent, and we expect it not only to furtherreduce the risk of solvent system toxicity, but also to minimize therisk that the organic solvent could potentiate clinical adverse effectsrelated to pimaricin.

It is apparent from the results that a dramatically improvedbioavailability of pimaricin was provided. Further, this novelpreparation yielded plasma drug concentrations and areas under theplasma concentration vs. time curves that were clearly fungicidal, basedon comparisons with our in vitro sensitivity studies with pimaricinagainst several strains of Aspergillus spp., and Candida spp., but mostimportantly against Fusarium spp., since this fungus is typicallymultidrug resistant. The present invention makes it feasible to obtainbeneficial effects of pimaricin against systemic mycoses, with thepotential for a major improvement in the outcome of such infections.

Compositions of the present invention can further include additionalpharmaceutically acceptable carriers, adjuvants, and/or biologicallyactive substances. Compositions of the present invention, as describedabove, can be used in methods for treatment or prophylaxis of systemicfungal infections in mammals, particularly in humans. The methodsinvolve administering to a mammal an amount of the compositionseffective to prevent, eliminate, or control the fungal infection. Theadministering step can suitably be parenteral (preferably by intravenousinjection). The compositions can also be administered intranasally as anaerosol. Such administration is preferably repeated on a timed schedule,and may be used in conjunction with other forms of therapy orprophylaxis, including methods involving administration of differentbiologically active agents to the subject. The dose administered of acomposition in accordance with the present invention is preferablybetween approximately 0.1 and 100 mg/kg of body weight of the mammaliansubject to which it is administered, most preferably between about 1-5mg/kg.

The preceding description of specific embodiments of the presentinvention is not intended to be a complete list of every possibleembodiment of the invention. Persons skilled in this field willrecognize that modifications can be made to the specific embodimentsdescribed here that would be within the scope of the present invention.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An antifungal composition, pharmaceuticallyacceptable for parenteral administration comprising:an amount ofpimaricin or a salt thereof that is effective to inhibit the growth of asystemic infection in a mammal; a pharmaceutically acceptable dipolaraprotic solvent; and a pharmaceutically acceptable aqueous secondarysolvent.
 2. The composition of claim 1, where the aprotic solvent isN,N-dimethylacetamide.
 3. The composition of claim 1, where the aqueoussecondary solvent is an aqueous lipid emulsion.
 4. The composition ofclaim 3, where the aqueous lipid emulsion comprises a lipid componentthat includes at least one vegetable oil and at least one fatty acid. 5.The composition of claim 4, where the lipid component comprises at leastabout 5% by weight soybean oil and at least about 50% by weight fattyacids.
 6. The composition of claim 1, where the secondary solvent iswater.
 7. The composition of claim 1, where the secondary solvent issaline solution.
 8. The composition of claim 1, where the secondarysolvent is dextrose solution.
 9. A method of preventing or treating asystemic infection in a mammal, comprising administering parenterally toa mammal a composition pharmaceutically acceptable for parenteraladministration that comprises:an amount of pimaricin or a salt thereofthat is effective to inhibit the growth of an infectious agent that issensitive to pimaricin in the mammal; a pharmaceutically acceptabledipolar aprotic solvent; and a pharmaceutically acceptable aqueoussecondary solvent.
 10. The method of claim 9, where the administrationis intravascular.
 11. The method of claim 9, where the aprotic solventis N,N-dimethylacetamide.
 12. The method of claim 9, where the aqueoussecondary solvent is an aqueous lipid emulsion.
 13. The method of claim12, where the aqueous lipid emulsion comprises a lipid component thatincludes at least one vegetable oil and at least one fatty acid.
 14. Themethod of claim 13, where the lipid component comprises at least about5% by weight soybean oil and at least about 50% by weight fatty acids.15. The method of claim 9, where the secondary solvent is selected fromthe group consisting of water, saline solution, and dextrose solution.16. An antifungal composition pharmaceutically acceptable for parenteraladministration, comprising:an amount of pimaricin or a salt thereofeffective to inhibit the growth of fungal infection in a mammal;N,N-dimethylacetamide; and an aqueous lipid emulsion that comprises atleast one vegetable oil and at least one fatty acid.
 17. A method ofpreventing or treating fungal infection in a mammal, comprising the stepof administering parenterally to a mammal a composition thatcomprises:an amount of pimaricin or a salt thereof effective to inhibitthe growth of fungal infection in a mammal; N,N-dimethylacetamide; andan aqueous lipid emulsion that comprises at least one vegetable oil andat least one fatty acid.